Biomaterials (v.29, #33)

Dentin non-collagenous matrix components (NCPs) are structural proteins involved in the formation, the architecture and the mineralization of the extracellular matrix (ECM). We investigated here how recombinant metalloproteinase stromelysin-1, also termed MMP-3, initiates the release of ECM molecules from artificially demineralized human dentin. Analysis of the supernatants by Western blotting reveals that MMP-3 extracts PGs (decorin, biglycan), and also a series of phosphorylated proteins: dentin sialoprotein (DSP), osteopontin (OPN), bone sialoprotein (BSP) and MEPE, but neither dentin matrix protein-1 (DMP1), another member of the SIBLING family, nor osteocalcin (OC), a non-phosphorylated matrix molecule. After treatment of dentin surfaces by MMP-3, scanning electron microscope (SEM) examination of resin replica shows an increased penetration of the resin into the dentin tubules when compared to surfaces only treated by demineralizing solutions. This preclinical investigation suggests that MMP-3 may be used to improve the adhesive properties of restorative materials.
Keywords: Dentin; Stromelysin-1 (MMP-3); Proteoglycans; Extracellular matrix proteins (ECM); Adhesion;

Self-assembled monolayers (SAMs) are being developed into coatings to reduce microbial biofilm formation on biomaterials. To test anti-adhesion properties, SAMs can be easily constructed on gold, and used to represent a coated biomaterial. However, coatings that prevent bacterial adhesion must also resist protein adsorption. We explored the competitive effects of bacteria and protein for adsorption to SAMs, choosing fetal bovine serum (FBS) to represent protein non-specific binding, and fibronectin (FN) to evaluate ligand/receptor binding. Staphylococcus epidermidis were immobilized on an atomic force microscope (AFM) tip and used as a force probe to detect the interaction forces between bacteria and gold-coated SAMs. The SAMs tested were alkanethiol molecules terminating in isophthalic acid (IPA) or isophthalic acid with silver (IAG). While S. epidermidis showed weak interactions with FBS, the bacteria showed strong adhesion with FN, due to ligand/receptor binding. Bacterial retention and viability experiments were correlated with the force measurements. S. epidermidis interacting with IAG SAMs showed a loss of viability, due to the mobility of silver ions. For most substrata, there was a link between high adhesion forces with bacteria and a high percentage of dead cells being retained on that substratum (even in the absence of a specific biocidal effect, such as silver). This may suggest that high adhesion forces can cause stress to the bacteria which contributed to their death. The relationship between highly adhesive SAMs and bacterial inactivation may be useful in future biomaterial design. When evaluating coatings for biomaterials, it is important to consider the interplay between bacteria, proteins, and the coating material.
Keywords: Bacterial adhesion; Atomic force microscopy; Adhesion; Biofouling;

Mechanisms of unmodified CdSe quantum dot-induced elevation of cytoplasmic calcium levels in primary cultures of rat hippocampal neurons by Mingliang Tang; Ming Wang; Tairan Xing; Jie Zeng; Huili Wang; Di-Yun Ruan (4383-4391).
Quantum dots (QDs) have shown great promise for applications in biology and medicine, which is being challenged by their potential nanotoxicity. Reactive oxygen species (ROS) produced by QDs are believed to be partially responsible for QD cytotoxicity. Cytoplasmic Ca2+ plays an important role in the development of ROS injury. Here we found unmodified cadmium selenium (CdSe) QDs could elevate cytoplasmic calcium levels ([Ca2+]i) in primary cultures of hippocampal neurons, involved both extracellular Ca2+ influx and internal Ca2+ release. More specifically, verapamil and mibefradil (L-type and T-type calcium channels antagonists, respectively) failed to prevent extracellular Ca2+ influx under QD insult, while ω-conotoxin (N-type antagonist) could partially block this Ca2+ influx. Surprisingly, this Ca2+ influx could be well blocked by voltage-gated sodium channels (VGSCs) antagonist, tetrodotoxin (TTX). QD-induced internal Ca2+ release could be avoided by clonazepam, a specific inhibitor of mitochondrial sodium–calcium exchangers (MNCX), and also by TTX. Furthermore, dantrolene, an antagonist of ryanodine (Ry) receptors in endoplasmic reticulum (ER), almost abolished internal Ca2+ release, while 2-APB [inositol triphosphate (IP3) receptors antagonist] failed to block this Ca2+ release, indicating that released Ca2+ from mitochondria, which was induced by extracellular Na+ influx, further triggered much more Ca2+ release from ER. Our results imply that more research on the biocompatibility and biosafety of QD is both warranted and necessary.
Keywords: Calcium overload; Hippocampus; Nanoparticles; QD; ROS;

The responses of osteoblasts, osteoclasts and endothelial cells to zirconium modified calcium-silicate-based ceramic by Yogambha Ramaswamy; Chengtie Wu; Annika Van Hummel; Valery Combes; Georges Grau; Hala Zreiqat (4392-4402).
In this study we have developed Ca3ZrSi2O9 (Baghdadite) ceramics by incorporating Zirconium in Ca–Si system and determined their biological properties. Ca3ZrSi2O9 ceramics possess apatite-formation ability in simulated body fluid, indicating their potential bioactivity. The response of human osteoblast like cells (HOB), osteoclast and endothelial cells when cultured on Ca3ZrSi2O9 ceramics was investigated. Scanning electron microscopy and immunofluorescence studies demonstrated that this material supports HOB cell attachment with organized cytoskeleton structure. Compared to CaSiO3, Ca3ZrSi2O9 ceramics induced increased HOB proliferation and differentiation as shown by increased methyltetrazidium salt (MTS), alkaline phosphatase activity, and mRNA expression levels of bone-related genes (Collagen type I, alkaline phosphatase, Bone Sialoprotein, receptor activator of NF-κB ligand and osteoprotegerin). Ca3ZrSi2O9 ceramics supported the fusion of monocytes to form functional osteoclasts with their characteristic features of f-actin ring structures and the expression of αvβ3 integrin consistent with functional activity. Osteoclasts cultured on Ca3ZrSi2O9 expressed increased levels of osteoclast-related genes; Cathepsin K, Carbonic Anhydrase II, Matrix metalloproteinase-9, receptor activator of NF-κB and Calcitonin Receptor, consistent with the formation of functional osteoclasts. In addition to HOB and osteoclasts, Ca3ZrSi2O9 supported the attachment of endothelial cells, which expressed the endothelial cell markers; ZO-1 and VE-Cadherin. Results presented here indicate that Ca3ZrSi2O9 ceramics have the potential for applications in bone tissue regeneration.
Keywords: Calcium silicate; Zirconium; Osteoblasts; Osteoclasts; Endothelial cells; Gene expression;

Surface immobilization of proteins provides various biomaterials that permit the control of cellular functions through protein–protein interactions. Our previous study demonstrated that human epidermal growth factor carrying a hexahistidine sequence at the C-terminus (hEGF-His) could be anchored to the Ni-chelated surface by coordination, providing the versatile substrate for the selective proliferation of neural stem cells. The present study was undertaken to gain deeper insights into the basis for such an outstanding property of the surface with coordinated hEGF-His. For this purpose, the structure of the coordinated hEGF-His was analyzed by multiple internal reflection-infrared absorption spectroscopy. In addition, stability of coordinate bonds was assessed under cell culture conditions using a spatially-restricted anchoring technique. These data were compared to the results obtained from surfaces with covalently immobilized and physically adsorbed hEGF-His. The results presented here demonstrate that coordinated hEGF-His remains its intact conformation and is firmly anchored to the surface during cell culture. These attributes are both crucial for establishing the adherent culture and hence selective expansion of neural stem cells.
Keywords: Epidermal growth factor; Stem cell; Surface immobilization; Recombinant protein; Patterning; Signaling;

Use of hybrid chitosan membranes and N1E-115 cells for promoting nerve regeneration in an axonotmesis rat model by S. Amado; M.J. Simões; P.A.S. Armada da Silva; A.L. Luís; Y. Shirosaki; M.A. Lopes; J.D. Santos; F. Fregnan; G. Gambarotta; S. Raimondo; M. Fornaro; A.P. Veloso; A.S.P. Varejão; A.C. Maurício; S. Geuna (4409-4419).
Many studies have been dedicated to the development of scaffolds for improving post-traumatic nerve regeneration. The goal of this study was to develop and test hybrid chitosan membranes to use in peripheral nerve reconstruction, either alone or enriched with N1E-115 neural cells. Hybrid chitosan membranes were tested in vitro, to assess their ability in supporting N1E-115 cell survival and differentiation, and in vivo to assess biocompatibility as well as to evaluate their effects on nerve fiber regeneration and functional recovery after a standardized rat sciatic nerve crush injury. Functional recovery was evaluated using the sciatic functional index (SFI), the static sciatic index (SSI), the extensor postural thrust (EPT), the withdrawal reflex latency (WRL) and ankle kinematics. Nerve fiber regeneration was assessed by quantitative stereological analysis and electron microscopy. All chitosan membranes showed good biocompatibility and proved to be a suitable substrate for plating the N1E-115 cellular system. By contrast, in vivo nerve regeneration assessment after crush injury showed that the freeze-dried chitosan type III, without N1E-115 cell addition, was the only type of membrane that significantly improved posttraumatic axonal regrowth and functional recovery. It can be thus suggested that local enwrapping with this type of chitosan membrane may represent an effective approach for the improvement of the clinical outcome in patients receiving peripheral nerve surgery.
Keywords: Nerve; Nerve regeneration; Nerve tissue engineering; Chitosan; Animal model;

The osteogenic differentiation of rat muscle-derived stem cells in vivo within in situ-forming chitosan scaffolds by Kyung Sook Kim; Jung Hwa Lee; Hyun Hee Ahn; Ju Young Lee; Gilson Khang; Bong Lee; Hai Bang Lee; Moon Suk Kim (4420-4428).
We herein examined the bone formation from rat muscle-derived stem cells (rMDSCs) using an injectable in situ-forming chitosan gel in vivo. The rMDSCs were easily isolated from rat muscle tissue. The osteogenic factors caused differentiation of rMDSCs toward the osteogenic lineage. The rMDSCs survived well on the scaffold created by the in vitro and in vivo in situ-forming chitosan gel, indicating that in situ gel-forming chitosan was a suitable substrate for the attachment and proliferation of rMDSCs. Bone formation was observed only in chitosan gel containing both rMDSCs and osteogenic factors. Subcutaneous implantation of the in situ-forming chitosan gel demonstrated that rMDSCs-containing chitosan gel induced much lower host tissue responses than did the chitosan gel alone, probably due to the immunosuppression of the transplanted rMDSCs.
Keywords: Rat muscle-derived stem cells; Osteogenic differentiation; Chitosan; In situ gel;

TAT-conjugated nanoparticles for the CNS delivery of anti-HIV drugs by Kavitha S. Rao; Maram K. Reddy; Jayme L. Horning; Vinod Labhasetwar (4429-4438).
We have shown that nanoparticles (NPs) conjugated to trans-activating transcriptor (TAT) peptide bypass the efflux action of P-glycoprotein and increase the transport of the encapsulated ritonavir, a protease inhibitor (PI), across the blood-brain-barrier (BBB) to the central nervous system (CNS). A steady increase in the drug parenchyma/capillary ratio over time without disrupting the BBB integrity suggests that TAT-conjugated NPs are first immobilized in the brain vasculature prior to their transport into parenchyma. Localization of NPs in the brain parenchyma was further confirmed with histological analysis of the brain sections. The brain drug level with conjugated NPs was 800-fold higher than that with drug in solution at two weeks. Drug clearance was seen within four weeks. In conclusion, TAT-conjugated NPs enhanced the CNS bioavailability of the encapsulated PI and maintained therapeutic drug levels in the brain for a sustained period that could be effective in reducing the viral load in the CNS, which acts as a reservoir for the replicating HIV-1 virus.
Keywords: Trans-activating transcriptor; Nanoparticles; Blood-brain-barrier; P-glycoprotein; Transport; Anti-HIV drugs;

A cardiomyocyte-targeted Fas siRNA delivery system was developed using prostaglandin E2 (PGE2)-modified siRNA polyplexes formed by a reducible poly(amido amine) to inhibit cardiomyocyte apoptosis. PGE2, which was used as a specific ligand for cardiomyocyte targeting, was conjugated to the terminal-end of the sense siRNA (PGE2-siRNA). The reducible cationic copolymer, synthesized via Michael-type polyaddition of 1,6-diaminohexane and cystamine bis-acrylamide (poly(DAH/CBA)), tightly condensed the PGE2-siRNA conjugate to form nanosize polyplexes having a diameter of 100–150 nm. The PGE2-siRNA/poly(DAH/CBA) polyplexes decomplexed to release PGE2-siRNA in a cytosolic reducing environment due to the degradation of the reducible poly(DAH/CBA). The cellular uptake of the PGE2-siRNA/poly(DAH/CBA) polyplex was increased in rat cardiomyocytes (H9C2 cells) due to PGE2 receptor-mediated endocytosis. When H9C2 cells were transfected with siRNA against Fas, a key regulator of ischemia-induced apoptosis, the PGE2–Fas siRNA/poly(DAH/CBA) polyplex delivery system led to a significant increase in Fas gene silencing, resulting in inhibition of cardiomyocyte apoptosis. The PGE2–Fas siRNA/poly(DAH/CBA) polyplex did not induce interferon-alpha in peripheral blood mononuclear cells. These results suggest that the PGE2–Fas siRNA/poly(DAH/CBA) polyplex formulation may be clinically applicable as a cardiomyocyte-targeted Fas siRNA delivery system to inhibit apoptosis in cardiovascular disease.
Keywords: Cardiomyocyte; siRNA; Fas; Prostaglandin E2; Reducible cationic polymer;

In this study, we investigated the utilization of highly sensitive immuno-PCR (IPCR) method as a powerful tool to detect NPC in early disease stage. We established a substrate-ELISA platform as a model system for evaluation of the feasibility of our idea after surface modification process on glass beads. Therein the DNA–antibody conjugation was added to sensitize prior enzyme substrate–antibody complex. In the study, the detection efficiency of two different systems regarding sensitivity, affinity, and specificity was evaluated. Moreover, to show the efficacy of our IPCR system, commercialized ELISA kit was also included for comparison with our IPCR glass substrate-based capture system. The surface physical properties of the modified substrates were also tested with atomic force microscopy and X-ray photoelectron spectroscopy, together with the measurement of the water contact angle. In the results, various factors in the production of IPCR detection system were determined to maximize the effect on assay performance, including the modification of the glass surface properties, primary and secondary antibody optimal concentrations, and biotinylated reporter DNA concentration. We found that the sensitivity of IPCR was approximately over two order magnitude higher than that of conventional ELISA method. The result suggests that our IPCR system could be an applicable and reliable tool for early detection of NPC.
Keywords: Surface modification; Silane; Immunoassay; Immuno-PCR; Cancer;

Changes in pore morphology and fluid transport in compressed articular cartilage and the implications for joint lubrication by George W. Greene; Bruno Zappone; Boxin Zhao; Olle Söderman; Daniel Topgaard; Gabriel Rata; Jacob N. Israelachvili (4455-4462).
Cartilage sections were cut from the middle zone of pig knee articular cartilage and attached to substrates in two different kinds of newly designed ‘pressure cells’, one for fluorescence the other for NMR measurements. The fluorescence cell was filled with buffer solution containing fluorescently marked 70 kDa dextran which was allowed to diffuse into the cartilage pores. A second glass surface was then pressed down onto the thin cartilage sample under different loads (pressures), and the resulting compression (strain) and change in pore volume were measured as a function of time, simultaneously with measurements of the lateral diffusion and flow pattern of the dextran molecules using Fluorescence Recovery After Photobleaching (FRAP). Complementary experiments were made on the normal diffusion coefficients of pure electrolyte solutions (no dextran) in thicker cartilage sections with pulse-gradient NMR using a new pressure cell suitable for such measurements. Taken together our results show that the highly anisotropic structure of cartilage has a strong effect on the way fluid diffuses laterally and normally at different stages of compression. Our results also show how geometric constraints on a cartilage network and trapped high MW polymer such as HA during normal compressions are likely to affect both the normal and the lateral mobilities of polyelectrolytes and water.
Keywords: Cartilage; Fluorescence recovery after photobleaching; FRAP; Pulse-gradient NMR; Collagen structure; Joint lubrication;

Molecular dynamics study of heparin based coatings by Martin Almlöf; Emma M.E. Kristensen; Hans Siegbahn; Johan Åqvist (4463-4469).
Heparin based surface coatings can be used to improve the biocompatibility of metallic surfaces such as vascular stents. Here, we report molecular dynamics simulations of a macromolecular conjugate of heparin used to prepare such surfaces. The structural properties of the heparin conjugate are investigated for different degrees of hydration, to allow comparison with spectroscopic results. The simulations show that the polymer becomes more compact with an increasing degree of inter-chain interactions as the hydration increases. This is also accompanied by changes in the interaction patterns among the heparin chains, where counter ions become looser associated with the disaccharide units and their strong interactions can be partly replaced by water molecules and heparin hydroxyl groups. The structural information that can be obtained from computer simulations of this type of coatings can be very valuable for understanding and further development of functional interfaces, since very little is known experimentally regarding their detailed structural properties.
Keywords: heparin coating; hydration; molecular dynamics simulation; photoelectron spectroscopy;